Summary This developmental project seeks to develop methods to dramatically boost immunogenicity of virus-like particle (VLP) vaccines. Ebola virus (EBOV) and Marburg virus (MARV) are members of the filovirus family of enveloped, negative-sense RNA viruses that causes outbreaks of severe human disease. Due to their lethality, filoviruses are among the handful of emerging viruses for which biosafety level 4 (BSL4) containment is required for their study. Although progress has been made toward EBOV vaccines, there remains a need for development of vaccines for other filoviruses, particularly MARV. VLPs are an often-explored vaccine platform for a variety of viruses, including for EBOV and MARV. Advantages of VLPs include their relative safety, as they cannot replicate and cause viral disease; their close resemblance to authentic virus, such that they present antigens in their native state; their capacity to elicit both B and T cell responses and their proven efficacy for a variety of systems. Potential challenges of VLPs as vaccines include relatively lower immunogenicity, as compared to live virus. Filovirus VLPs can be produced by co-expression of the viral matrix protein VP40, the viral glycoprotein (GP), with or without expression of the viral nucleoprotein (NP). Such VLPs have been demonstrated to stimulate dendritic cell (DC) responses, to elicit B and T cell mediated immunity and to protect mice, guinea pigs and non- human primates (NHPs) from lethal challenge. Therefore, filovirus VLPs are viable vaccine candidates. However, filovirus VLP immunogenicity is weak with protection requiring coadministration with an adjuvant and multiple doses. This is not ideal for vaccines that are mainly deployed in response to outbreaks. This application seeks to dramatically improve immunogenicity of EBOV and MARV VLPs by the rational incorporation of type I interferon (IFN) signaling domains from the cellular pattern recognition receptors RIG-I or TRIF into the particles. This enables delivery into cells of the IFN inducing domain by the VLPs, triggering robust innate immune responses reminiscent of live virus infection, to boost adaptive immune responses. To further pursue this direction, we will explore approaches to optimize the incorporation of IFN-inducing signaling domains into EBOV and MARV VLPs. We will then evaluate the capacity of the different enhanced VLP strategies to elicit B and T cell immune responses in vivo, and finally, test the most promising enhanced VLPs for the capacity to elicit rapid protection against EBOV and MARV challenge in mice. Successful completion of these studies will provide a novel platform for the development of filovirus vaccines and an approach that can likely be applied to other emerging viral pathogens.